Antibody-Drug Conjugates and Cellular Metabolic Dynamics

Description

This book summarizes the related research achievements in Antibody-drug conjugates (ADCs) and their cell metabolism kinetics. The book has three main parts. The first part describes the basic theory of ADCs, including the basic concept and structure of ADCs, and the relationship between the targets of ADCs and their specific functions. The second part mainly introduces the endocytosis and intracellular metabolism of ADCs, including the relationship between endocytosis and ADC activity, the endocytosis and intracellular transport of ADCs, the distribution and metabolism of ADC in vivo. Then it discusses the new formats and research technology of ADCs, including the application of miniaturized antibodies in ADC synthesis, novel carriers for ADC design, the technology and application of site-specific conjugation, and approaches for analyzing the drug: antibody ratio (DAR), the study of pharmacokinetics of ADCs. This book combines the basic theory with the research technology. It can be used as a reference book for students, teachers and researchers of biomedical field. Preface Contents Chapter 1: Antibody-Drug Conjugates: Basic Concepts and Structures 1.1 Basic Concepts 1.2 Monoclonal Antibodies Used for ADC Development 1.3 Linkers 1.4 Effector Molecules 1.4.1 Tubulin Inhibitors 1.4.2 DNA-Damaging Antibiotics 1.5 Challenges and Prospective References Chapter 2: Relationship Between Target and Specific Action of Antibody-Drug Conjugates 2.1 Introduction 2.2 Tumor Associated Antigens 2.2.1 Membrane Proteins Associated with Cell Proliferation 2.2.2 Carcinoembryonic Antigen 2.2.3 Leukocyte Differentiation Antigen 2.3 Tumor Specific Antigen 2.3.1 Tumor Specific pMHC 2.3.1.1 Tumor-Specific pMHC Under Investigation as Targets of Antibody Drugs 2.3.1.2 Tumor-Specific pMHC as Potential Targets of Antibody Drugs 2.3.2 Tumor Specific Mutant Extracellular Domain of Membrane Protein 2.4 Conclusion References Chapter 3: The Internalization and Therapeutic Activity of Antibody Drug Conjugates 3.1 Introduction 3.2 Experimental Steps for Evaluating Internalization 3.2.1 Materials 3.2.2 Internalization Assay by Flow Cytometry 3.2.3 Intracellular Localization Assay by Confocal Microscope 3.3 Strategies to Improve Internalization Efficiency 3.3.1 Identifying Antibody Binding Epitopes 3.3.2 Antibody Drug Conjugate Toxins 3.3.3 Application of Bispecific Antibody 3.3.4 Application of Biparatopic Antibody 3.4 Conclusion References Chapter 4: The Internalization and Intracellular Trafficking of ADCs 4.1 Introduction 4.2 The Endocytosis Pathways of ADC 4.2.1 Clathrin Mediated Endocytosis (CME) 4.2.2 Caveolae Mediated Endocytosis (CavME) 4.2.3 Macropinocytosis 4.3 Intracellular Trafficking of ADC 4.3.1 Early Endosome (EE) 4.3.2 Late Endosome (LE) 4.3.3 Lysosome 4.4 Drug Release 4.5 Methods in Tracing the Intracellular Transport of ADC 4.5.1 Flow Cytometry Analysis to Analyze Endocytosis 4.5.2 Confocal Microscopy to Monitor Internalization and Intracellular Trafficking of ADC 4.5.3 The Colocalization of Internalized ADC and Lysosome 4.6 Conclusions References Chapter 5: Distribution and Metabolism of Antibody-Drug Conjugates 5.1 Preface 5.2 The Mechanism of Action of ADC in Cells 5.2.1 The Release of Cytotoxic Payloads 5.2.2 Mechanism of Action and Metabolism of Cytotoxic Payloads 5.2.3 Metabolism of Antibody Parts in ADCs 5.3 Methods and Procedures in Studying Distribution and Metabolism of ADC 5.3.1 Ligand Binding Assay 5.3.2 Liquid Chromatography-Tandem Mass Spectrometry Assay 5.3.3 Radiolabeled Antibody Assay 5.4 Summary References Chapter 6: Application of Antibody Fragments in ADCs 6.1 Introduction 6.2 Antibody Fragment Drug Conjugates (AFDCs) 6.2.1 Materials 6.2.2 Methods 6.2.2.1 Expression and Purification of Fab and Fab-CH3mut Miniaturized Antibodies 6.2.2.2 Preparation of Fab-vcMMAE and Fab-CH3mut-vcMMAE 6.2.2.3 Affinity of Fab, Fab-CH3mut, OFA, Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMAME 6.2.2.4 In Vitro Anti-Tumor Activity of Fab-vcMMAE, Fab-CH3mut-vcMMAE, OFA-vcMMAE 6.2.2.5 Apoptosis-Inducing Activity of Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMMAE on Tumor Cell 6.2.2.6 In Vivo Biodistribution of Fab, Fab-CH3mut and OFA 6.2.2.7 Antitumor Activity and Toxicity of Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMMAE in Vivo 6.3 Overview of Antibody Fragments 6.4 Summary References Chapter 7: Novel Targeting Carriers in Antibody-Drug Conjugates 7.1 Introduction 7.2 The Feasibility of TRAIL as a Conjugate Carrier 7.2.1 TRAIL and its Receptor 7.2.2 Cell Apoptosis Inducing and Anti-Tumor Activity of TRAIL 7.2.3 Study on TRAIL as a Cytotoxin Carrier 7.2.3.1 Materials 7.2.3.2 Experiments [28] 7.3 Review Study on Other Novel Carriers 7.4 Summary References Chapter 8: Site-Specified Conjugating Technology and Application 8.1 Introduction 8.1.1 Engineered Cysteine 8.1.2 Insertion of Unnatural Amino Acids 8.1.3 Enzyme-Chemical Conjugation 8.2 Site-Specified Conjugation of Mutated Cysteine 8.2.1 Principle of Site-Specified Conjugation of Mutated Cysteine 8.2.2 Main Experimental Materials 8.2.3 Main Experimental Procedures [20] (DAR = 2 or DAR = 4) 8.2.3.1 Acquisition of Cysteine Mutant Antibodies 8.2.3.2 Synthesis and Analysis of Ab-vcMMAE 8.3 Site-Specific Conjugation Through Sortase A 8.3.1 Direct Enzymatic Approach 8.3.1.1 Materials 8.3.1.2 Methods 8.3.2 Chemo-Enzymatic (Srt A) Strategy 8.3.2.1 Materials 8.3.2.2 Methods[24] 8.4 Identification of Conjugation Sites by Mass Spectrometry 8.4.1 Materials 8.4.2 Methods[25] 8.5 Conclusion References Chapter 9: Determination of Drug-to-Antibody Ratio of ADCs 9.1 Introduction 9.2 Experimental Procedures for RP-HPLC and HIC to Determine DAR 9.2.1 Determination of DAR by RP-HPLC 9.2.1.1 Experimental Conditions and Methods 9.2.1.2 Data Processing 9.2.2 Determination of DAR by HIC 9.2.2.1 Experimental Conditions and Methods 9.2.2.2 Data Processing 9.2.3 Case Analysis [18] 9.3 The Experimental Procedure for the Determination of DAR by LC-MS 9.3.1 Instruments and Reagents 9.3.1.1 Instruments 9.3.1.2 Reagents 9.3.2 Method 9.3.2.1 Sample Preparation 9.3.2.2 LC-MS Analysis 9.3.2.3 Data Processing 9.3.3 Case Analysis [18] 9.3.3.1 Relative Molecular Mass of Light and Heavy Chains 9.3.3.2 Peptide Map Detection 9.3.3.3 Results 9.4 Conclusion References Chapter 10: Pharmacokinetic Study of Antibody-Drug Conjugates 10.1 Introduction 10.2 The Application of ELISA-Based Methods in Studying Pharmacokinetic of ADC 10.2.1 ELISA-Sandwich Technique 10.2.1.1 Total Antibody Detection 10.2.1.2 Total ADC Detection 10.2.1.3 Active ADC Detection 10.2.2 Ligand Binding Assay Based on ELISA 10.2.2.1 Detection of Active Antibody 10.2.2.2 Detection of Active ADC 10.2.3 Semihomogeneous Assay 10.2.3.1 Specific SHA 10.2.3.2 Non-specific SHA 10.3 The Application of Flow Cytometry in Studying Pharmacokinetics of Antibody-Drug Conjugates 10.3.1 Making Biotinylated Anti-MMAE Antibody 10.3.2 Quantitative OFA-HL and OFA-MMAE with Flow Cytometry Method 10.3.3 The Lower Limit of Quantification Is Affected by Selection of Fluorescein 10.3.4 Quantification of OFA-HL and OFA-HL-MMAE in Blood Samples 10.4 Summary References